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Abstract

Multifunctional porous Si nanostructure is designed to optically monitor enzymatic
activity of horseradish peroxidase. First, an oxidized PSi optical nanostructure,
a Fabry-Pérot thin film, is synthesized and is used as the optical transducer element.
Immobilization of the enzyme onto the nanostructure is performed through DNA-directed
immobilization. Preliminary studies demonstrate high enzymatic activity levels of
the immobilized horseradish peroxidase, while maintaining its specificity. The catalytic
activity of the enzymes immobilized within the porous nanostructure is monitored in
real time by reflective interferometric Fourier transform spectroscopy. We show that
we can easily regenerate the surface for consecutive biosensing analysis by mild dehybridization
conditions.

Keywords:

Porous Si; DNA immobilization; Enzyme; Nanostructure; Biosensor

Background

Nanostructured porous Si (PSi) has emerged as a promising material for optical biosensing
applications due to its large internal surface area and tunable optical properties
[1-3]. Numerous biosensing applications, including the detection of DNA hybridization
[4], proteins
[5,6], and enzymatic activity
[7,8], have been presented, demonstrating the advantages of these nanosystems in terms
of improved detection sensitivity, label-free and real-time rapid analysis. However,
the major challenge in designing these biosensors arises from the intrinsic instability
of the recognition element during the immobilization procedures onto the transducer’s
surface
[9-11].

The present work describes a highly versatile approach for reversible enzyme conjugation
to a porous SiO2 (PSiO2) surface via DNA-directed immobilization (DDI). This approach has been successfully
used for the immobilization of different classes of enzymes onto a range of solid
supports, without affecting their biological activity
[9,12]. DDI strategy utilizes the site-selectivity and the high affinity of Watson-Crick
base pairing between two complimentary strands. Taking into account that DNA hybridization
is a reversible process, the anchored enzyme as well as the biosensor surface can
be regenerated and reused for multiple reaction cycles. Herein, we demonstrate this
concept for a model enzyme, horseradish peroxidase (HRP), which is one of the most
active peroxidases and often used as a powerful tool in biotechnology (e.g., organic
synthesis, immune-detection, and wastewater treatment)
[13,14].

Methods

Preparation of porous SiO2 nanostructures

Single side polished on the <100 > face oriented and heavily doped p-type Si wafers
(0.8 to 1.0 mΩ·cm resistivity, B-doped, from Siltronix Corp., Archamps, France) are
electrochemically etched in a 3:1 (v/v) solution of aqueous HF (48%, Merck, Whitehouse Station, NJ, USA) and ethanol (99.9%,
Merck) at a constant current density of 385 mA cm−2 for 30 s. Si wafers with an exposed area of 1.33 cm2 are contacted on the backside with a strip of aluminum foil and mounted in a Teflon
etching cell; a platinum mesh is used as the counterelectrode. After etching, the
surface of the wafer is rinsed with ethanol several times and dried under a dry nitrogen
gas. The freshly-etched PSi samples are thermally oxidized in a tube furnace (Thermolyne)
at 800°C for 1 h in ambient air, resulting in a PSiO2 layer.

Biofunctionalization of PSiO2

The PSiO2 films are first immersed in a solution of 3-glycidoxypropyl(trimethoxy)silane (GPTS)
dissolved in toluene (90 mM) for 2 h. The samples are then extensively rinsed with
toluene, ethanol, and acetone and dried under a nitrogen gas. The sequence of the
capture strand is 5′-amino TCCTGTGTGAAATTGTTATACGCC-3′ (aD1). A solution of the amino-modified
probe (100 μM) is applied onto the silanized PSiO2 surface and incubated for 2 h in a humidity chamber, followed by a post-cleaning
process. Afterwards, 2.6-μM of the complimentary strand (cD1)-modifiedHRP conjugates
are incubated onto the PSiO2 for 30 min. Finally, the PSiO2 samples are rinsed with 50-mM PBS, soaked in the buffer for 20 min, and vigorously
rinsed again with PBS to remove any unbounded species from the surface. All processes
are carried out at a room temperature (RT), below the melting temperature of the DNA
(65.6°C). Mild basic conditions are used for dissociation of the hybridized complex
(0.1-M NaOH for 5 min) to allow surface regeneration.

Fluorescent labeling and fluorescence microscopy

Fluorescently labeled (5′-TAMRA-modified) complimentary strand (TAMRA-cD1, 1 μM) is
used to confirm DNA hybridization onto the modified PSiO2 surface Following the introduction the fluorescently tagged strand, the PSiO2 samples are characterized using TIRF iMIC microscope, and images are taken using
interline transfer CCD (Andor Clara E, Till Photonics, Munich, Germany) and fast filter-based
illumination system with 488 nm TIRF laser beam or the oligochrome light. Data are
analyzed by LA live acquisition software.

Optical measurements

Interferometric reflectance spectra of PSiO2 samples are collected using an Ocean Optics CCD USB 4000 spectrometer (Dunedin, FL,
USA) fitted with a microscope objective lens coupled to a bifurcated fiber optic cable.
A tungsten light source was focused onto the center of the sample surface with a spot
size approximately 1 to 2 mm
[2]. Reflectivity data are recorded in the wavelength range of 400 to 1,000 nm, with
a spectral acquisition time of 100 ms. Both illumination of the surface and detection
of the reflected light are performed along an axis coincident with the surface normal.
All the optical experiments are conducted in a fixed cell in order to assure that
the sample’s reflectivity is measured at the same spot during all the measurements.
All optical measurements are collected in aqueous surrounding. Spectra are collected
using a CCD spectrometer and analyzed by applying fast Fourier transform, as previously
described
[15,16].

Enzymatic activity of HRP

Ampliflu Red stock solutions are prepared according to the manufacturer’s instructions
using 50-mM PBS buffer (pH 7.4). Final concentrations of 1-mM H2O2 and 0.1-mM Ampliflu Red are used to characterize the HRP activity levels
[9]. Solutions of 200 μL are placed on the HRP-modified PSiO2 samples, and the reaction progress is monitored for 8 min at RT. The fluorescence
values of the reaction product resorufin are recorded at 590 nm, using an excitation
wavelength of 530 nm.

The relative activity is calculated by the following relationship:

(1)

Optical sensing of HRP activity

The PSiO2 is washed with 50-mM PBS buffer solution (pH 7.4) for 30 min. Then, 0.8-mM of 4-chloro-1-naphthol
in PBS buffer is introduced and continually cycled through the flow cell for 20 min.
Finally, 0.16-M H2O2 is added to the cycled solution for HRP surface activation. The reflectivity spectra
of the sample are recorded throughout the experiment.

Results and discussion

Biofunctionalization of PSiO2 with DNA-enzyme conjugates

The PSiO2 surfaces are prepared from a highly doped p-type single-crystal Si wafer, polished
on the <100 > face using an anodic electrochemical etch. The resulting nanostructure
is then thermally oxidized to generate a stable and a more hydrophilic scaffold
[17]. The structural properties are as previously described by Massad-Ivanir et al.
[16]. Briefly, the porous layer is 7,880 ± 60 nm thick with interconnecting cylindrical
pores ranging in diameter from 60 to 100 nm, and the calculated porosity is approximately
80%. The semisynthetic approach for anchoring DNA-protein conjugates onto the PSiO2 surface is based on a well-established methodology of DNA-directed immobilization
to solid surfaces, e.g., gold, glass, and polymers
[9,12]. The detailed synthesis is schematically outlined in Figure
1a. First, the thermally oxidized nanostructure is epoxy-silanized with GPTS to obtain
an activated surface for grafting of amine-modified single-stranded capture DNA (aD1).
The complimentary thiolated oligonucleotide strand (cD1) is cross-linked to the HRP
using sulfo-SMCC bi-functional linker containing NHS and maleimide moieties
[9]. In the final step, the resulting DNA-enzyme (HRP-cD1) conjugates are hybridized
with the capture probe to enable their immobilization onto the PSiO2 surface. Hybridization is confirmed by studying the annealing of a fluorescently
labeled complimentary DNA strand to the aD1-modified PSiO2. Figure
1b depicts a fluorescence microscope image of aD1-modified PSiO2 surface following exposure to the TAMRA-cD1, revealing a large number of small fluorescent
regions on the PSiO2 surface. In a control experiment, the silanization step is omitted from the conjugation
scheme, and the PSiO2 is exposed to TAMRA-cD1 only. In this case, no florescence signal is observed (Figure
1c), confirming that there is no or negligible interaction of the TAMRA-cD1 with PSiO2 surface in the absence of the immobilized aD1. Thus, these experiments demonstrate
that the presence of the complementary capture strand is necessary for the immobilization
of the TAMRA-cD1 onto the biosensing platform.

Figure 1.Conjugation of enzymes to a porous SiO2surface using DDI approach.(a) A schematic representation of the synthetic steps required for enzyme immobilization
onto PSiO2 through DDI approach. The porous scaffold is first modified with GPTS to graft the
amine-modified capture strand (aD1) that serves as an anchor for the following hybridization
process. (b) Results of fluorescence labeling experiments to confirm the specificity of the DDI
method. Complete bioconjugation; aD1 is attached to the silane-modified porous surface
followed by hybridization of the complementary strand labeled with TAMRA fluorophore.
(c) Control experiment; no GPTS is used (silanization step).

Reflective interferometric Fourier transform spectroscopy

Reflective interferometric Fourier transform spectroscopy (RIFTS) is used as a complimentary
tool to monitor the optical changes occurring during the biofunctionalization steps
onto of the PSiO2 surface. This method presents high sensitivity to small changes in the average refractive
index of the porous thin film, allowing for direct and real-time monitoring the binding
of different species to the pore walls
[1,3,5,7,8]. The reflectivity spectrum of the thin PSiO2 film consists of a series of interference fringes that result from a Fabry-Pérot
interference. This fringe pattern arises from reflections at the top and at the bottom
of the film so that the measurement is made over the entire volume of the system.
The effective optical thickness (EOT) refers to the 2 nL term in the Fabry-Pérot formula (where n is the average refractive index; L, the thickness of the film; and λ, the wavelength of the incident light)
[15]. A change in the average refractive index leads to a shift in the observed reflectivity
spectrum that correlates with EOT changes. It is expected that the chemical modification
of the porous nanostructure (as previously outlined in Figure
1a) will result in a redshift of the EOT due to the increase in the average refractive
index upon attachment of different species to the pore walls. It should be noted that
the nanostructure is designed to allow proper infiltration of the DNA conjugates (approximate
size of 10 nm
[18]) into the pores, characterized by an average diameter of 80 nm. Indeed, significant
EOT changes are observed after each of the described immobilization steps, see Figure
2a. Following the grafting of aD1 to the epoxy-functionalized PSiO2, an EOT increase of 49 ± 1 nm is detected. DDI of the HRP-cD1 conjugates (230 ng
in 2 μL volume) causes an additional EOT increase of 85 ± 17 nm. Additionally, the
specificity of the hybridization is validated by addition of non-modified HRP to the
aD1-modified PSiO2, resulting in insignificant EOT changes (Figure
2b). Thus, the RIFTS results further confirm that the enzyme immobilization onto the
PSiO2 surface occurs through Watson-Crick base pairing.

Enzymatic activity assays

The enzymatic activity of the anchored enzymes onto the porous scaffold is studied
by monitoring the oxidation products of HRP (Figure
3d). The modified PSiO2 samples are exposed to a substrate solution of Ampliflu Red in PBS, and the collected
solution is spectrophotometrically analyzed. Figure
3b depicts the high relative activity (74%) of the immobilized conjugates compared
to non-modified HRP activity in a solution (Equation 1). Thus, the modified enzymes
retain their specificity and activity once anchored and confined on the porous support.
An important advantage of the DDI anchoring is the ability to regenerate the surface,
as schematically shown in Figure
3a, by applying mild dehybridization conditions, e.g., pH change or temperature increase
(above the melting temperature of the DNA pair). Indeed, following a mild-base wash,
an insignificant relative enzymatic activity value of less than 3% is attained (Figure
3c), indicating that the PSiO2 surface can be regenerated. In addition, a consecutive cycle of hybridization/dehybridization
is carried out to demonstrate the reversibility of DDI method for the characterization
of small aliquots (2 μL) of the recovered enzyme and its reuse. These experiments
show the high reversibility and reproducibility of the immobilization strategy as
relative activity values of 49% and less than 3% are recorded, followed by DDI and
dehybridization processes, respectively (Figure
3b,c).

Figure 3.Reversible HRP immobilization onto porous SiO2surface using DDI and the corresponding enzymatic activity.(a) A schematic illustration of reversible enzyme immobilization onto PSiO2 surface using DDI approach. Note: These schematics are for illustration purposes
only as all modifications occur also inside the pores. (b) Relative activity of HRP immobilized via DDI onto PSiO2. (c) Removal of the DNA-enzyme conjugates form the surface by mild dehybridization. Two
consecutive cycles of DNA-enzyme hybridization/dehybridization are performed. (d) HRP enzymatic activity is quantified using the Ampliflu Red assay in which oxidation
of the non-fluorescence Ampliflu substrate occurs in the presence of HRP and H2O2 to fluorescence resorufin.

Optical detection of enzymatic activity

Previous sections have demonstrated that the DNA-HRP conjugates remain catalytically
active after the DDI onto the PSiO2 nanostructure. Next, we investigated the potential of this platform to act as an
optical biosensor for monitoring enzymatic activity through RIFTS experiments. Thus,
HRP-modified PSiO2 is fixed in a flow cell setup and is exposed to various substrate/buffer combinations.
The reactions are monitored in real time by acquisition of the reflectivity spectra
of the porous thin film. The recorded EOT values prove a direct measure of the amount
of enzymatic activity products infiltrating the pores, as previously shown by Orosco
et al.
[7]. Figure
4 depicts the EOT change of the HRP-modified nanostructure following the introduction
of 4-chloro-1-naphthol, which oxidizes in the presence of peroxidases into insoluble
4-chloro-1-naphton
[19] (Figure
4, inset). A rapid increase of 90 nm in the EOT value is observed after injection of
0.16-M H2O2 to the cycled solution (Figure
4b). The EOT increase is attributed to infiltration and accumulation of the oxidation
products in the pores as the enzymatic reaction occurs
[20]. Control experiments with bare and only aD1-modified PSiO2 (no immobilized enzyme) exposed to the same conditions exhibit insignificant changes
in EOT (data not shown), indicating that the biosensor platform is indeed specific
for monitoring the enzymatic activity products.

Figure 4.Optical response of HRP-modified porous SiO2to enzymatic reaction products infiltrating into the nanostructure. The HRP-modified PSiO2 nanostructure is pretreated with 50-mM PBS (pH 7.4) to minimize nonspecific adsorption
of proteins. (a) Wash with 0.8-mM 4-chloro-1-naphthol in PBS buffer (pH 7.4). (b) Addition of H2O2 to the cycled solution. The PSiO2 biosensor is fixed in a custom-made flowcell, and the reflectivity spectra are recorded
every 30 s. Inset: oxidation of 4-chloro-1-naphthol in the presence of HRP and H2O2.

Conclusions

We demonstrate that DNA-directed protein immobilization is an elegant and facile method
for specific and reversible anchoring of a model enzyme, i.e., HRP onto PSiO2 Fabry-Pérot thin films. HRP specific immobilization through Watson-Crick base pairing
onto the porous nanostructure is confirmed by both fluorescence microscopy and RIFTS.
The surface-immobilized HRP retains relatively high enzymatic activity in comparison
to its activity in solution. We show that the catalytic activity of the HRP immobilized
within the PSiO2 thin film can be monitored in real time by RIFTS technique. For biosensor design,
the DDI method allows the use of small enzyme quantities for monitoring the reaction
while allowing both enzyme and surface to be regenerated for subsequent usage. This
‘proof-of-concept’ biosensor scheme can be potentially extended for systematic analysis
of the enzyme of interest under unlimited experimental setups.

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

GS carried out PSi synthesis, enzyme immobilization, enzyme activity assays, biosensing
experiments, and data analysis. He also wrote the manuscript. NMI contributed to the
conception of the experiments and was involved in the synthesis and characterization
of PSi. SE synthesized the DNA-enzyme conjugates. MS participated in the design of
the study. LF conceived the study and coordinated the synthesis and characterization
of DNA-enzyme conjugates. ES conceived and coordinated the study, participated in
the design of the study and data analysis, and drafted the manuscript. All authors
read and approved the final manuscript.

Acknowledgments

ES acknowledges the financial support of the NEVET program administered by the Russell
Berrie Nanotechnology Institute and by the Marie Curie Reintegration Grant (NANOPACK)
administered by the European Community. GS acknowledges the Miriam and Aaron Gutwirth
Excellence Scholarship Award. This work was also supported by Weizmann-Technion-KIT
Start up grant and DFG-CFN Excellence Initiative Project A5.7.